Purification of tissue plasminogen activator (tPA)

ABSTRACT

A method is disclosed for obtaining affinity ligands for isolating tissue-type plasminogen activator (tPA). Ligands binding tPA with high specificity at pH 7 and releasing tPA at pH 5 or lower are disclosed. Also disclosed are methods whereby additional ligands having desirable preselected binding and release (elution) characteristics may be isolated, permitting the development of tailored ligands to meet the purification problems presented by any particular feed stream containing tPA.

[0001] This is a continuation-in-part of copending U.S. application Ser.No. 08/619,885 filed Mar. 20, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of proteinpurification. Specifically, the present invention relates to discoveryof and isolation of affinity ligands useful for the purification oftissue plasminogen activator, or tPA.

BACKGROUND OF THE INVENTION

[0003] Human tissue-type plasminogen activator, or tPA, is a proteolyticenzyme produced by endothelial cells which has high affinity for fibrincontained in aggregates of coagulated blood (i.e., clots, or “thrombi”).tPA also serves to activate and convert plasminogen, an inactiveproenzyme, into plasmin, a thrombolytic enzyme. Since tPA binds tofibrin in thrombi and activates plasminogen there to dissolve clots, tPAhas become an important drug for use as a thrombolytic.

[0004] Although tPA has become a leading drug in the treatment ofthrombosis, it competes against other effective thrombolytic agents,such as streptokinase and urokinase, which are arguably less effectivebut cost much less. In order for tPA to remain among the most-prescribedthrombolytic agents or to be distributed to even greater numbers ofpatients, ways in which tPA can be produced more efficiently or at lowercost must be explored.

[0005] Effective means for eliminating impurities such as cell debris,pathogens, non-human proteins, etc. from a production feed stream isalso important in the production of tPA, as it is with any proteinproduct intended ultimately for therapeutic administration to humanpatients.

[0006] Thus, there is a continuing need for the development of improvedreagents, materials and techniques for the isolation of tPA on a moreefficient and cost-effective basis.

[0007] Affinity chromatography is a very powerful technique forachieving dramatic single-step increases in purity. Narayanan (1994),for instance, reported a 3000fold increase in purity through a singleaffinity chromatography step.

[0008] Affinity chromatography is not, however, a commonly usedtechnique in large-scale production of biomolecules. The ideal affinitychromatography ligand must, at acceptable cost, (1) capture the targetbiomolecule with high affinity, high capacity, high specificity, andhigh selectivity, (2) either not capture or allow differential elutionof other species (impurities); (3) allow controlled release of thetarget under conditions that preserve (i.e., do not degrade or denature)the target; (4) permit sanitization and reuse of the chromatographymatrix; and (5) permit elimination or inactivation of any pathogens.However, finding high-affinity ligands of acceptable cost that cantolerate the cleaning and sanitization protocols required inpharmaceutical manufacturing has proved difficult (see, Knight, 1990).

[0009] Murine monoclonal antibodies (MAbs) have been used effectively asaffinity ligands. Monoclonal antibodies, on the other hand, areexpensive to produce, and they are prone to leaching and degradationunder the cleaning and sanitization procedures associated withpurification of biomolecules, leading MAb-based affinity matrices tolose activity quickly (see, Narayanan, 1994; Boschetti, 1994). Inaddition, although MAbs can be highly specific for a target, thespecificity is often not sufficient to avoid capture of impurities thatare closely related to the target. Moreover, the binding characteristicsof MAbs are determined by the immunoglobulin repertoire of the immunizedanimal, and therefore practitioners must settle for the bindingcharacteristics they are dealt by the animal's immune system, i.e.,there is little opportunity to optimize or select for particular bindingor elution characteristics using only MAb technology. Finally, themolecular mass per binding site (25 kDa to 75 kDa) of MAbs and even MAbfragments is quite high.

[0010] Up until now, there have been no known affinity ligands suitablefor the purification of tPA that approach the characteristics of theideal affinity ligand described above, that not only bind to the targettPA molecule with high affinity but also release the tPA under desirableor selected conditions, that are able to discriminate between the tPAand other components of the solution in which the tPA is presented,and/or that are able to endure cleaning and sanitization procedures toprovide regenerable, reusable chromatographic matrices.

[0011] Such tPA affinity ligands and methods for obtaining them areprovided herein.

SUMMARY OF THE INVENTION

[0012] The present invention provides methods for obtaining affinityligands for tPA which exhibit desirable or selected binding propertiesand release properties. The affinity ligands of the present inventionexhibit not only favorable binding characteristics for affinityseparation of tPA but also desired release (elution) characteristics andother desired properties such as stability, resistance to degradation,durability, reusability, and ease of manufacture.

[0013] The tPA affinity ligands of the present invention may beinitially identified from a peptide library, such as a phage displaylibrary, by a method comprising:

[0014] (a) selecting a first solution condition (i.e., the bindingconditions) at which it is desired that an affinity ligand should bindto the tPA;

[0015] (b) selecting a second solution condition (i.e., the releaseconditions) at which it is desired that an affinity complex between thetPA and the affinity ligand will dissociate, wherein the second solutioncondition is different from the first solution condition;

[0016] (c) providing a library of analogues of a candidate bindingdomain, wherein each analogue differs from said candidate binding domainby variation of the amino acid sequence at one or more amino acidpositions within the domain;

[0017] (d) contacting said library of analogues with tPA at the firstsolution condition, for sufficient time to permit analogue/tPA bindingcomplexes to form;

[0018] (e) removing analogues that do not bind under the first solutioncondition;

[0019] (f) altering the conditions of the solution of contacting step(e) to the second solution condition; and

[0020] (g) recovering the candidate binding analogues released under thesecond solution condition, wherein the recovered analogues identifyisolated tPA affinity ligands.

[0021] Following this general procedure, several polypeptide affinityligands for tPA have been isolated, as described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows a chromatogram of tissue plasminogen activator (25 μLof 1 mg/mL tPA) over an affinity chromatography column having animmobilized tPA affinity ligand (CMTI derivative #109, described inExample 1), with elution over a pH 7-pH 3 gradient. The peak at 15minutes is estimated to contain approximately 90% of the injected tPA.

[0023]FIG. 2 shows a chromatogram of Coagulation Standard (diluted 10×)over a tPA affinity column (CMTI derivative #109) with elution asdescribed above. The small peak at 15.6 minutes was shown to be agradient artifact.

[0024]FIG. 3 shows a chromatogram of a mixture consisting of 25 μL ofCoagulation Standard (Diluted 10×) spiked with 25 μL of tPA, withelution over a pH 7-pH 3 gradient. The peak at 1 minute is thecollection of plasma proteins and the peak at 15.4 minutes is the tPA.

[0025]FIG. 4 shows the same chromatogram as shown in FIG. 3 withvertical scale expanded.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention makes possible the efficient purificationof tPA by affinity chromatography.

[0027] The tPA may be produced in any known way, including chemicalsynthesis; production in transformed host cells; secretion into culturemedium by naturally occurring or recombinantly transformed bacteria,yeasts, fungi, insect cells, and mammalian cells; secretion fromgenetically engineered organisms (e.g., transgenic mammals); or inbiological fluids or tissues such as urine, blood, milk etc. Thesolution that contains the crude tPA as it is initially produced (i.e.,the production solution) will sometimes be referred to as the “feedstream”.

[0028] Each method of producing tPA yields tPA in a feed stream thatadditionally contains a number of impurities (with respect to tPA). Onepurpose of the present invention is to produce affinity ligands andpreparations (such as chromatography media) comprising such ligands thatallow rapid and highly specific purification of tPA from any feedstream. The tPA affinity ligands obtained herein are tailored to theisolation of tPA from a particular feed stream, under specificpreselected conditions. If an alternate production method for the tPA isused, producing a different feed stream, a different set of affinityligands may be necessary to achieve the same level of purification. Thenew set of ligands can be readily obtained by following the proceduresoutlined herein.

[0029] tPA affinity ligands of the invention bind the tPA to the virtualexclusion of any other molecule in the feed stream with high affinity.Further, the affinity ligands release the tPA intact and in active formwhen the solution conditions are changed.

[0030] Selecting Binding and Release Conditions

[0031] In order to isolate new affinity ligands for tPA, two solutionconditions are selected, i.e., binding conditions and releaseconditions. The binding conditions are a set of solution conditionsunder which it is desired that a discovered affinity ligand will bindthe target tPA; the release conditions are a set of solutions conditionsunder which it is desired that a discovered affinity ligand will notbind the tPA. The two conditions may be selected to satisfy anycriterion of the practitioner, such as ease of attaining the conditions,compatability with other purification steps, lowered cost of switchingbetween conditions compared to other affinity media, etc. Preferably thetwo solution conditions are (a) well within the boundaries of thestability envelope for the tPA and (b) far apart with respect to atleast one solution parameter. For example, if the tPA is stable over awide pH range, then favorable binding conditions might be pH 7.5, 150 mMsalt, 25° C. and favorable release conditions might be pH 3, 150 mMsalt, 25° C. For a different tPA form having a narrow range of pHstability (for example, pH 6.2 to 7.8) but being stable over a widerange of salinity, two useful conditions might be binding conditions: pH7.2, 3 M NaCl, 25° C. and release conditions: pH 7.2, 2 mM NaCl, 25° C.

[0032] Selection of a Candidate Binding Domain

[0033] In conjunction with selecting specific solution conditions forthe desired binding and release of the tPA, a candidate binding domainis selected to serve as a structural template for the engineeredaffinity ligands that will exhibit the desired binding and releasecapabilities. The binding domain may be a naturally occurring orsynthetic protein, or a region or domain of a protein. The candidatebinding domain may be selected based on knowledge of a known interactionbetween the candidate binding domain and the tPA, but this is notcritical. In fact, it is not essential that the candidate binding domainhave any affinity for tPA at all: Its purpose is to provide a structurefrom which a multiplicity (library) of analogues can be generated, whichmultiplicity of analogues will include one or more analogues thatexhibit the desired binding and release properties (and any otherproperties selected for). Thus, the binding conditions and the releaseconditions discussed infra may be selected with knowledge of the exactpolypeptide that will serve as the candidate binding domain, or withknowledge of a class of proteins or domains to which the candidatebinding domain belongs, or completely independently of the choice of thecandidate binding domain. Similarly, the binding and/or releaseconditions may be selected with regard to known interactions between abinding domain and the tPA, e.g., to favor the interaction under one orboth of the solution conditions, or they may be selected without regardto such known interactions. Likewise, the candidate binding domain canbe selected taking into account the binding and/or release conditions ornot, although it must be recognized that if the binding domain analoguesare unstable under the binding or release conditions no useful affinityligands will be obtained.

[0034] In selecting a candidate binding domain, the object is to providea template or parental structure from which a library of similarlystructured analogue domains can be generated. The analogue library willpreferably be a biased library (as opposed to a randomly generatedlibrary), in that variegation of the basic domain to create the librarywill be carried out in such a way as to favor the properties desired forthe affinity ligands.

[0035] The nature of the candidate binding domain greatly influences theproperties of the derived proteins (analogues) that will be testedagainst the tPA molecule. In selecting the candidate binding domain, themost important consideration is how the analogue domains will bepresented to the tPA, i.e., in what conformation the tPA and theanalogues will come into contact. In preferred embodiments, for example,the analogues will be generated by insertion of synthetic DNA encodingthe analogue into a replicable genetic package, resulting in display ofthe domain on the surface of a microorganism, such as M13 phage, usingtechniques as described, e.g., in U.S. Pat. No. 5,403,484 (Ladner etal.) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporated herein byreference.

[0036] Structured polypeptides offer many advantages as candidatebinding domains over unstructured peptides. Mutation of surface residuesin a protein will usually have little effect on the overall structure orgeneral properties (such as size, stability, temperature ofdenaturation) of the protein; while at the same time mutation of surfaceresidues may profoundly affect the binding properties of the protein.This has been fully documented, for example, for BPTI-homologous Kunitzdomains (see Ladner, 1995). Mutating surface residues on proteins orstructured domains can lead to greater diversity of properties for theanalogues than is obtained by mutating unstructured peptides because theprotein framework or the structure of the domain holds the mutatedresidues in conformations that differ from residue to residue and fromframework to framework. This is especially important for hydrophobicside groups that would become buried unless constrained in a structure.The more tightly a peptide segment (domain) is constrained, the lesslikely it is to bind to any particular target. If it does bind, however,the binding is likely to be tighter and more specific. Thus, it ispreferred to select a candidate binding domain and, in turn, a structurefor the peptide analogues, that is constrained within a framework havingsome degree of rigidity. Alternatively, more than one candidate bindingdomain structure can be selected, in order to increase the size of thelibrary of analogues and to introduce additional variegated structuresfor presentation to the tPA. As the size of the library is increased,and higher numbers and diversely structured analogues are prepared, theprobability of including a useful framework and displayed functionalgroups increases to the point where high-affinity ligands can be foundfor isolation of the tPA from virtually any feed stream.

[0037] The size of the candidate binding domain is also an importantconsideration. Small proteins or polypeptides offer several advantagesover large proteins, such as monoclonal antibodies (see Ladner, 1995).First, the mass per binding site is reduced. Highly-stable proteindomains having low molecular weights, e.g., Kunitz domains (˜7 kDa),Kazal domains (˜7 kDa), Cucurbida maxima trypsin inhibitor (CMTI)domains (˜3.5 kDa), and endothelin (˜2 kDa), can show much higherbinding per gram than do antibodies (150 kDa) or single-chain antibodies(30 kDa).

[0038] Second, the possibility of non-specific binding is reducedbecause there is less surface available.

[0039] Third, small proteins or polypeptides can be engineered to haveunique tethering sites in a way that is impracticable for antibodies.For example, small proteins can be engineered to have lysines only atsites suitable for tethering (e.g., to a chromatography matrix), butthis is not feasible for antibodies. It is often found that only a smallfraction of immobilized antibodies are active, possibly due toinappropriate linkage to the support.

[0040] Most small proteins or polypeptides that are stabilized bydisulfides do not contain cysteines that are not involved in disulfides.This is because the oxidizing conditions that cause disulfide formationfor stabilizing the protein also lead to disulfide formation byotherwise unpaired cysteines. Thus, small proteins having stabilizingdisulfides and an odd number of cysteines tend to form disulfide linkeddimers (e.g., homodimers or heterodimers). The disulfides betweendomains are more easily reduced than are the stabilizing intradomaindisulfides. Thus, by selective reduction it is possible to obtainmonomeric disulfide-stabilized domains having a single free thiol. Suchthiols can be used for highly stable immobilization of these domains byformation of a thioether with iodoaceamide, iodoacetic acid, or similarα-iodo carboxylic acid groups.

[0041] Small protein or polypeptide domains also can be chemicallysynthesized, which permits special immobilizing groups to beincorporated in a way that does not interfere with binding to the tPA.For instance, if small disulfide-containing proteins are chemicallysynthesized, the amino acid sequence can be altered by adding an extracysteine residue with the thiol blocked in a different manner from othercysteines elsewhere in the sequence. The selected thiols can bedeblocked and disulfides allowed to form, then the added cysteine can bedeblocked and the molecule can be immobilized by reaction with asuitable material such as a substrate containing immobilizedNH₂—CO—CH₂I.

[0042] Fourth, a constrained polypeptide structure is more likely toretain its functionality when transferred with the structural domainintact from one framework to another. For instance, the binding domainstructure is likely to be transferable from the framework used forpresentation in a library (e.g., displayed on a phage) to an isolatedprotein removed from the presentation framework or immobilized on achromatographic substrate.

[0043] There are many small, stable protein domains suitable for use ascandidate binding domains and for which the following useful informationis available: (1) amino acid sequence, (2) sequences of severalhomologous domains, (3) 3-dimensional structure, and (4) stability dataover a range of pH temperature, salinity, organic solvent, oxidantconcentration. Some examples are: Kunitz domains (58 amino acides, 3disulfide bonds), Cucurbida maxima trypsin inhibitor domains (31 aminoacids, 3 disulfide bonds), domains related to guanylin (14 amino acids,2 disulfide bonds), domains related to heat-stable enterotoxin IA fromgram negative bacteria (18 amino acids, 3 disulfide bonds), EGF domains(50 amino acids, 3 disulfide bonds), kringle domains (60 amino acids, 3disulfide bonds), fungal carbohydrate-binding domains (35 amino acids, 2disulfide bonds), endothelin domains (18 amino acids, 2 disulfidebonds), and Streptococcal G IgG-binding domain (35 amino acids, nodisulfide bonds). Most, but not all of these contain disulfide bondsthat rigidify and stabilize the structure. Libraries based on each ofthese domains, preferably displayed on phage or other genetic packages,can be readily constructed and used for the selection of bindinganalogues.

[0044] Providing a Library of Candidate Binding Domain Analogues

[0045] Once a candidate binding domain has been selected, a library ofpotential affinity ligands is created for screening against the tPA atthe binding and elution (release) conditions. The library is created bymaking a series of analogues, each analogue corresponding to thecandidate binding domain except having one or more amino acidsubstitutions in the sequence of the domain. The amino acidsubstitutions are expected to alter the binding properties of the domainwithout significantly altering its structure, at least for mostsubstitutions. It is preferred that the amino acid positions that areselected for variation (variable amino acid positions) will be surfaceamino acid positions, that is, positions in the amino acid sequence ofthe domains which, when the domain is in its most stable conformation,appear on the outer surface of the domain (i.e., the surface exposed tosolution). Most preferably the amino acid positions to be varied will beadjacent or close together, so as to maximize the effect ofsubstitutions. In addition, extra amino acids can be added into thestructure of the candidate binding domain. In preferred embodiments,especially where a great deal of information is available concerning theinteractions of the tPA with other molecules, particularly the candidatebinding domain, those amino acid positions that are essential to bindinginteractions will be determined and conserved in the process of buildingthe analogue library (i.e., the amino acids essential for binding willnot be varied).

[0046] The object of creating the analogue library is to provide a greatnumber of potential affinity ligands for reaction with the tPA molecule,and in general the greater the number of analogues in the library, thegreater the likelihood that a member of the library will bind to the tPAand release under the preselected conditions desired for release. On theother hand, random substitution at only six positions in an amino acidsequence provides over 60 million analogues, which is a library sizethat begins to present practical limitations even when utilizingscreening techniques as powerful as phage display. It is thereforepreferred to create a biased library, in which the amino acid positionsdesignated for variation are considered so as to maximize the effect ofsubstitution on the binding characteristics of the analogue, and theamino acid residues allowed or planned for use in substitutions arelimited to those that are likely to cause the analogue to be responsiveto the change in solution conditions from the binding conditions to therelease conditions.

[0047] As indicated previously, the techniques discussed in U.S. Pat.No. 5,223,409 are particularly useful in preparing a library ofanalogues corresponding to a selected candidate binding domain, whichanalogues will be presented in a form suitable for large-scale screeningof large numbers of analogues with respect to a target tPA molecule. Theuse of replicable genetic packages, and most preferably phage display,is a powerful method of generating novel polypeptide binding entitiesthat involves introducing a novel DNA segment into the genome of abacteriophage (or other amplifiable genetic package) so that thepolypeptide encoded by the novel DNA appears on the surface of thephage. When the novel DNA contains sequence diversity, then eachrecipient phage displays one variant of the initial (or “parental”)amino acid sequence encoded by the DNA, and the phage population(library) displays a vast number of different but related amino acidsequences.

[0048] A phage library is contacted with and allowed to bind the tPAmolecule, and non-binders are separated from binders. In various ways,the bound phage are liberated from the tPA and amplified. Since thephage can be amplified through infection of bacterial cells, even a fewbinding phage are sufficient to reveal the gene sequence that encodes abinding entity. Using these techniques it is possible to recover abinding phage that is about 1 in 20 million in the population. One ormore libraries, displaying 10-20 million or more potential bindingpolypeptides each, can be rapidly screened to find high-affinity tPAligands. When the selection process works, the diversity of thepopulation falls with each round until only good binders remain, i.e.,the process converges. Typically, a phage display library will containseveral closely related binders (10 to 50 binders out of 10 million).Indications of convergence include increased binding (measured by phagetiters) and recovery of closely related sequences. After a first set ofbinding polypeptides is identified, the sequence information can be usedto design other libraries biased for members having additional desiredproperties, e.g., discrimination between tPA and particular fragments.

[0049] Such techniques make it possible not only to screen a largenumber of analogues but make it practical to repeat the binding/elutioncycles and to build secondary, biased libraries for screeninganalog-displaying packages that meet initial criteria. Thus, it is mostpreferred in the practice of the present invention (1) that a library ofbinding domain analogues is made so as to be displayed on replicablegenetic packages, such as phage; (2) that the library is screened forgenetic packages binding to tPA wherein the binding conditions of thescreening procedure are the same as the binding conditions preselectedfor the desired affinity ligand; (3) that genetic packages are obtainedby elution under the release conditions preselected for the affinityligand and are propagated; (4) that additional genetic packages areobtained by elution under highly disruptive conditions (such as, e.g.,pH 2 or lower, 8 M urea, or saturated guanidinium thiocyanate, toovercome extremely high affinity associations between some displayedbinding domain analogues and the target tPA) and are propagated; (5)that the propagated genetic packages obtained in (3) or (4) areseparately or in combination cycled through steps (2) and (3) or (4) forone or more additional cycles; and (6) a consensus sequence ofhigh-affinity binders is determined for analogues expressed in geneticpackages recovered from such cycles; (7) that an additional biasedlibrary is constructed based on the original framework (candidatebinding domain) and allowing the high-affinity consensus at eachvariable amino acid position, and in addition allowing other amino acidtypes selected to include amino acids believed to be particularlysensitive to the change between the binding conditions and the releaseconditions; (8) that this biased library is screened for members that(a) bind tightly (i.e., with high affinity) under the binding conditionsand (b) release cleanly (i.e., readily dissociate from the tPA target)under the release conditions.

[0050] Use of the Affinity Ligands in Chromatography

[0051] After members of one or more libraries are isolated that bind toa tPA with desired affinity under binding conditions and release fromthe tPA as desired under release conditions, isolation of the affinityligands can be accomplished in known ways. If, for example, the analoguelibrary is composed of prospective affinity ligands expressed on phage,released phage can be recovered, propagated, the synthetic DNA insertencoding the analogue isolated and amplified, the DNA sequence analyzedand any desired quantity of the ligand prepared, e.g., by directsynthesis of the polypeptide or recombinant expression of the isolatedDNA or an equivalent coding sequence.

[0052] Additional desired properties for the ligand can be engineeredinto an analogue ligand in the same way release properties wereengineered into the ligand, by following similar steps as describedabove.

[0053] The affinity ligands thus isolated will be extremely useful forisolation of tPA by affinity chromatography methods. Any conventionalmethod of chromatography may be employed. Preferably, an affinity ligandof the invention will be immobilized on a solid support suitable, e.g.,for packing a chromatography column. The immobilized affinity ligand canthen be loaded or contacted with a feed stream under conditionsfavorable to formation of ligand/tPA complexes, non-binding materialscan be washed away, then the tPA can be eluted under conditions favoringrelease of the tPA molecule from a ligand/tPA complex. Alternatively,bulk chromatography can be carried out by adding a feed stream and anappropriately tagged affinity ligand together in a reaction vessel, thenisolating complexes of the tPA and ligand by making use of the tag(e.g., a polyHis affinity tag, which can by used to bind the ligandafter complexes have formed), and finally releasing the tPA from thecomplex after unbound materials have been eliminated.

[0054] It should be noted that although precise binding and releaseproperties are engineered into the affinity ligands, subsequent use inaffinity purification may reveal more optimal binding and releaseconditions under which the same isolated affinity ligand will operate.Thus, it is not critical that the affinity ligand, after isolationaccording to this invention, be always employed only at the binding andrelease conditions that led to its separation from the library.

[0055] Finally, it should be kept in mind that the highest affinityligand is not necessarily the best for controllable or cost-effectiverecovery of a tPA molecule. The method of the invention permitsselection of ligands that have a variety of desirable characteristsimportant to the practitioner seeking isolation of tPA from a particularfeed steam, such as specific binding of the tPA coupled with predictableand controlled, clean release of the tPA, useful loading capacity,acceptably complete elution, re-usability/recyclability, etc.

[0056] Isolation of tPA affinity ligands in accordance with thisinvention will be further illustrated below. The specific parametersincluded in the following examples are intended to illustrate thepractice of the invention, and they are not presented to in any waylimit the scope of the invention.

EXAMPLE 1

[0057] The techniques described above were employed to isolate affinityligands for recombinant human tissue-type plasminogen activator (tPA).The process of creating tPA affinity ligands involved three generalsteps: (1) screening of approximately 11 million variants of a stableparental protein domain for binding to tPA, (2) producing smallquantities of the most interesting ligands, and (3) chromatographictesting of one ligand bound to activated beads for the affinitypurification of tPA from a plasma spiked sample.

[0058] For this work, tPA was purchased from CalBiochem (#612200) andimmobilized on ReactiGel™ agarose beads from Pierce Chemical Company bymethods described in Markland et al. (1996). Approximately 200 μg of tPAwere coupled to 200 μL of ReactiGel™ slurry.

[0059] Four libraries of phage-displayed proteins were picked for thescreening process. Three were based on the first Kunitz domain oflipoprotein associated coagulation inhibitor (LACI-K1), called Lib#1,Lib#3, and Lib#5 and one was based on Cucurbida maxima trypsin inhibitorI (CMTI-I). CMTI-I is a protein found in squash seeds and is able towithstand the acidic and proteolytic conditions of the gut. Theseproteins each have three disulfide bridges, making them highlyconstrained and stable. Members of these protein families have beenshown to have outstanding thermal stability (>80° C. without loss ofactivity), outstanding pH stability (no loss of activity on overnightincubation at pH 2 and 37° C. or on 1 hour exposure to pH 12 at 37° C.),and outstanding stability to oxidation. The number of potential aminoacid sequences in each library is given in Table 1 below: TABLE 1 Phagedisplay library populations used in the tPA screening Library nameparental domain Number of members Lib#1 LACI-K1    31,600 Lib#3 LACI-K1  516,000 Lib#5 LACI-K1  1,000,000 CMTI CMTI-I  9,500,000 Total11,000,000

[0060] The total diversity of the phage-display libraries screenedagainst tPA in this work is estimated to be around 11 million. TheKunitz domain and the CMTI domain could display much greater diversityby varying other parts of their surfaces.

[0061] Two screening protocols were used: “slow screen” and “quickscreen”. In a slow screen, phage from each round were amplified in E.coli before the next round. In a quick screen, phage recovered from thetPA in one round served as the input for the next round withoutamplification. In a quick screen, both the input and recovered number ofphage decreased rapidly over several rounds. The input level can be keptconstant in a slow screen. The constant input in a slow screen allowscomparisons between rounds that can indicate selection or lack thereof,but comparisons between rounds of quick screens are difficult tointerpret. Quick screening increases the likelihood that phage will beselected for binding rather than other irrelevant properties (e.g.,infectivity or growth rates).

[0062] The phage libraries described were screened for binding to tPAthrough four rounds. In the first round, the phage libraries were mixedin separate reactions with tPA agarose beads at pH 7 inphosphate-buffered saline (PBS). Bovine serum albumin (BSA) was added at0.1% to reduce non-specific binding. Unbound phage were washed off at pH7, and the bound phage eluted at pH 2 for the first screen only. Thesubsequent three quick screens had a different elution protocol and usedpooled outputs of the first screen. Pool A consisted of the combinedoutputs from the CMTI and Lib#1 libraries, and Pool B consisted of thecombined outputs of the Lib#3 and Lib#5 libraries. The binding of pooledlibraries was performed at pH 7, however, the first elution to removebound phage was carried out at pH 5 and a subsequent elution at pH 2 toelute phage that are released in the pH 5-pH 2 range. This was repeatedtwice more for a total of 4 rounds of selection.

[0063] The phage titers from the final three rounds of screening areshown below in Table 2. The output of one round was the input to thenext round. TABLE 2 Phage titers prior to screening and after the lastthree rounds of screening libraries against tPA Pool A Pool A Pool BPool B pH 5 elution pH 2 elution pH 5 elution pH 2 elution Prior toQuick 7 × 10¹¹ 7 × 10¹¹ 5 × 10¹¹ 5 × 10¹¹ Screening After second 1 ×10⁸  3 × 10⁷  7 × 10⁶  3 × 10⁶  round After third 2 × 10⁵  2 × 10⁶  2 ×10³  7 × 10³  round After fourth 3 × 10⁴  2 × 10⁵  150 90 round

[0064] From the phage titers, it is appears that Pool A converged andcontains strong binders, whereas Pool B had neither significantconvergence nor strong binders.

[0065] Forty phage clones were selected from the third round quickscreen selectants of each pool for further analysis, 20 from the pH 5pool and 20 from the pH 2 pool. The phage DNA was amplified using PCR todetermine whether CMTI- or LACI-derived gene fragments were present.

[0066] CMTI-derived constructs were found in 38 out of 40 phage isolatesfrom the quick screen of pool A. The remaining isolates did not yield aPCR product, indicating a deletion. Only 10 of the 40 phage isolatesfrom the quick screen of pool B contained the appropriate construct,another indication that the search had not succeeded.

[0067] One sign that a particular phage-displayed protein has a highaffinity for the tPA molecule is that it is found repeatedly. From the18 CMTI-derived phage isolates that release at pH 2, one sequence wasfound five times, a second, four times, and two of the remainingoccurred three times. The 18 sequences formed a closely-related familyof selected molecules, a further sign that the search had successfullyconverged.

[0068] Table 3 shows the variability of the observed sequences as afunction of the permitted variability and the selection pH. The CMTIlibrary was constructed by introducing combinatorial sequence diversityinto codons specifying a surface-exposed loop formed between cysteines 3and 10 of the parental CMTI protein. The cysteines were not variedbecause they form an important part of the structure. TABLE 3Construction of CMTI Library by Variegation of CMTI-I LSWP FSYC FSYCamino acids QRM LPHR LPHR encoded TKVVA ITNV I TNV (SEQ ID NO: 2) F Y SG A R EG C ADG KRTI ADG codon position −5 −4 −3 −2 −1 1 2 3 4 5 6 codonsTTC TAT TCC GGA GCC CGT NNG TGT NNT ANA NNT (SEQ ID NO: 3) restrictionsites P3 P2 P1 P1′ or position FSYC LSWP amino acids LLPHR QRM encodedITNV TKVA (SEQ ID NO: 2) ADG EG EKRG C K K D S D C L codon position 7 89 10 11 12 13 14 15 16 17 codons NNT NNG RRG TGT AAG AAG GAT TCT GAT TGCTTA (SEQ ID NO: 3) restriction sites P2′ P3′ or position amino acidsencoded (SEQ ID NO: 2) A E C V C L E H G Y C codon position 18 19 20 2122 23 24 25 26 27 28 codons GCA GAA TGC GTT TGC CTC GAG CAT GGT TAT TGT(SEQ ID NO: 3) restriction sites or position amino acids encoded (SEQ IDNO: 2) G A G P S Y I E G R I codon position 29 100 101 102 103 104 105106 107 108 109 codons GGC GCC GGT CCT TCA TAC ATT GAA GGT CGT ATT (SEQID NO: 3) restriction sites or position amino acids encoded (SEQ ID NO:2) V G S A A E . . . rest of mature III codon position 110 111 112 113201 202 codons GTC GGT AGC GCC GCT GAA . . . rest of coding sequence(SEQ ID NO: 3) for III restriction sites or position

[0069] Table 3 shows the DNA sequence of the CMTI library. Residues F⁻⁵and Y⁻⁴ correspond to residues 14 and 15 in the signal sequence ofM13mp18 from which the recipient phage was engineered. Cleavage bySignal Peptidase I (SP-I) is assumed to occur between A₁ and R₁.Residues designated 100-113 make up a linker between the CMTI variantsand mature III, which begins with residue A₂₀₁. The amino acid sequenceY₁₀₄IEGRIV should allow specific cleavage of the linker with bovineFactor X_(a) between R₁₀₈ and I₁₀₉. The M13-related phage in which thislibrary was constructed carries an ampicillin-resistance gene (Ap^(R))so that cells infected by library phage become Ap resistant. At eachvariable amino acid position, the wild-type amino acid residue is shownunderscrored. The amino acid sequence shown in Table 3 is designated SEQID NO: 2; the nucleotide sequence shown in Table 3 is designated SEQ IDNO: 3.

[0070] The isolates obtained from the pH 5 selection procedure exhibitedgreater sequence diversity than did the pH 2 selectants (Table 4).Despite the greater sequence variability, pH 5 selectants comprised afamily of closely-related protein sequences. Forming all combinations ofthe amino-acid types observed at each position gives only 13,400(=2×4×3×4×7×5×4) which is 0.15% of the original population. In the 20sequences determined, there were four sequences that occurred more thanonce, suggest that the actual diversity is less than 13,400. Althoughthe family of pH 5-selected sequences is clearly related to the pH2-selected family, there was only one example of sequence identitybetween the two sequence populations. TABLE 4 Reduction in variabilityat positions in CMTI upon selection for binding to tPA Position: 2 3 4 56 7 8 9 10 Permitted 13 C 15 4 15 15 13 4 C Variability Variability 2 C4 3 4 7 5 4 C of pH 5 Selectants Variability 1 C 1 2 3 3 1 2 C of pH 2Selectants

[0071] At positions 6 and 7, most (12 of 15) allowed amino acid typeswere rejected in the pH 2 selectants. From the selected sequences, it isnot clear whether the selected amino acids at positions 6 and 7contributed to binding or merely represent the elimination ofunacceptable possibilities at these positions.

[0072] The powerful convergence of the selection process is particularlyevident for the pH 2 selectants at positions 2, 4 and 8, where, althoughmany amino acid types could occur, only one amino acid type was found.This is a strong indication that this specific amino acid is critical tobinding. At each of these positions, the uniquely selected type of thepH 2 population was also the most common type at that position in the pH5 population. Allowing all observed amino acid types at each position ofthe pH 2 pool gives only 36 sequences, 0.0004% of the initialpopulation. That several sequences appeared more than once suggests thenumber of different sequences present in the pH 2 pool is not largerthan 36.

[0073] Table 5 shows the amino acid sequences of the variegated region(amino acid positions 1-12) for the 38 sequenced analogues of CMTI-I.The appearance of methionine residues at a position not designed to bevaried (position 11) indicates a DNA synthesis error in formation of thelibrary. TABLE 5 AMINO ACID SEQ position 1 2 3 4 5 6 7 8 9 10 11 12 IDCMTI-I R V C P R I L M E C K K 1 101 R W C P K T S L G C M K 4 102 R L CP K T Y L G C M K 5 103 R W C S T Y S L G C M K 6 104 R W C S T Y S L GC M K 7 105 R L C P K T S L E C M K 8 106 R W C S T Y S L G C M K 9 107R L C P K T S L E C M K 10 108 R W C S K S S L E C M K 11 109 R L C P KT D L G C M K 12 110 R W C P K S S M G C K K 13 111 R W C P R T V Q E CM K 14 112 R W C P T A P L E C M K 15 113 R L C P K T D L G C M K 16 114R W C P K S A L D C K K 17 115 R W C T K T S R E C M K 18 116 R W C I RT D L G C M K 19 117 R W C P K T S L G C M K 20 118 R W C P R T V R R CM K 21 119 R W C P K T H K E C M K 22 120 R W C P K T S L E C M K 23 221R W C P K S T L G C M K 24 222 R W C P K S T L G C M K 25 223 R W C P KY T L E C M K 26 224 R W C P R S S L E C M K 27 225 R W C P R Y T L E CM K 28 226 R W C P R S N L E C M K 29 227 R W C P K S N L E C M K 30 228R W C P K Y T L E C M K 31 229 R W C P K Y T L E C M K 32 230 R W C P RY T L E C M K 33 231 R W C P K S T L E C M K 34 232 R W C P K T S L G CM K 35 233 R W C P K Y T L E C M K 36 234 R W C P R S S L E C M K 37 235R W C P K S T L G C M K 38 236 R W C P R S S L E C M K 39 237 R W C P RS N L E C M K 40 238 R W C P R S N L E C M K 41

[0074] In Table 5, analogues having the sequences designated 101-120were obtained by elution at pH 5 and analogues having the sequences221-238 were obtained by fractionation at pH 2.

[0075] The specificity of phage-bound ligand candidates was tested bydetermining their affinity for other immobilized proteins. The phagebound proteins showed no affinity for the related human serum proteasesplasmin and thrombin bound to beads (data not shown). Additionally,experiments were performed on the phage isolates to determine relativeaffinity for and release characteristics from immobilized tPA.

[0076] In the case of the pH 5-releasing phage isolates, the majority ofthe phage are released at pH 5 and an order of magnitude fewer arereleased by further dropping the pH to 2. This indicates suitableaffinity ligands with a relatively dean release upon lowering the pH to5. In the case of the pH 2 releasing isolates, only isolate #232 gave atruly selective binding at pH 5 and then release at pH 2.

[0077] Ligand Synthesis and Immobilization

[0078] Next, free CMTI-derivative polypeptides were synthesized usingthe sequence information determined from the DNA of the phage isolates.Although the CMTI derivatives (analogues) could have been readilychemically synthesized, it was decided to express the polypeptides inyeast. One of the pH 5-releasing isolates, #109 (Table 5; ref. SEQ IDNO. 12), and one of the pH 2-releasing isolates, #232 (Table 5; ref. SEQID NO. 35), were selected for expression in Pichia pastoris. Atpositions 4 and 8, isolate #109 has the same amino acid types as seer inthe pH 2 selectants; at position 2, isolate #109 differs from the pH 2selectants.

[0079] The appropriate gene constructs were synthesized and insertedinto the Pichia pastoris expression system (Vedvick et al., 1991 andWagner et al., 1992) to create production strains. Five-literfermentations of each strain resulted in high-level expression. It wasestimated that the proteins were secreted into the fermentation broth inexcess of 1 g/L. The crude fermentation suspensions were clarified bycentrifugation, and 0.2 μ microfiltration steps. The 0.2 μ filtrateswere purified by ultrafiltration using PTTK cassettes with a 30 kDa NMWLcutoff. The ligands were purified from the ultrafiltration filtrates bycation exchange chromatography with Macro-Prep High S cation exchangesupport (BioRad), followed by two reversed-phase separations. Thereversed-phase separations used a linear gradient starting with watercontaining 0.1% TFA and having increasing acetonitrile (containing 0.1 %TFA) which was increased to 50% at 90 minutes. The resulting protein wasmore than 95% pure as measured by PDA spectral analysis on a 5 μmreversed phase column.

[0080] The ligand candidate was immobilized on abis-acrylamide/azlactone copolymer support with immobilizeddiaminopropylamine (Emphaze Ultralink™, Pierce Chemical Co.) accordingto the manufacturer's instructions. About 30 mg of CMTI analogue #109were coupled to 1 mL of the activated chromatography support.

[0081] Column Testing

[0082] A Waters AP Minicolumn, 5.0 cm×0.5 cm ID nominal dimensions wasmodified by the addition of a second flow adaptor which allowed thecolumn length to be reduced to 2.5 cm. This column was packed with#109-Emphaze Utralink™ beads using the recommended protocol and washedusing a series of increasing NaCl concentration washes at pH 7concluding with a 1 M wash.

[0083] In a first test of the #109 affinity column tissue-typeplasminogen activator obtained from CalBiochem was made up tomanufacturer's specifications to provide a 1 mg/mL solution of tPA.Coagulation Standard (Coagulation Control Level 1 from SigmaDiagnostics, Catalog #C-7916) lyophilized human plasma, wasreconstituted according to the manufacturer's instructions, then diluted10× and the tPA added. This sample was loaded onto the column and elutedin the presence of 1 M NaCl in all buffers, which was sufficient tosuppress non-specific protein binding to the column and to permit thepH-controlled binding and release of the tPA. There were two distinctpeaks: The first contained the plasma proteins (which were not retainedon the column); the second, obtained after lowering the pH, containedthe tPA, without any Madrid plasma components. The results wereconfirmed by silver stained gel (not shown). 90% of the tPA product wasrecovered.

[0084] A second affinity column using the #109 CMTI analogue wasprepared using EAH Sepharose 4B™ agarose beads (Pharmacia; Upsala SE) asthe chromatography support. The separation was performed on an HPLCsystem manufactured by Waters Inc. (Milford, Mass.). The systemcomprised a Model 718 Autoinjector, a Model 600 solvent delivery systemwith pumpheads capable of delivering 20 mL/minute, and a Model 996photodiode array detector. All of the equipment was installed accordingto manufacturer's specifications. The system was controlled by a Pentium133 IBM-compatible computer supplied by Dell Corp. The computer wasfurnished with a 1 gigabyte hard drive, 16 megabytes of RAM, and a colormonitor, onto which the Millenium software supplied by Waters Inc. wasloaded.

[0085] Spectral data in the range 200 nm to 300 nm were collected with1.2 nm resolution. FIGS. 1, 2, 3, and 4 were collected at 280 nm. Themobile phases for the chromatographic work were Buffer A and Buffer B:Buffer A consisted of 25mM potassium phosphate, 50 mM arginine, and 125mM NaCl, buffered to pH 7 with potassium hydroxide. Buffer B consistedof 50 mM potassium phosphate and 150 mM NaCl, buffered to pH 3 withphosphoric acid. In all cases, samples were injected in 100% Buffer A,followed by washing with 100% Buffer A from t=0 to t=2 min. From t=2min. to t=8 min., elution was with 100% Buffer B. After t=8 min.,elution was with 100% Buffer A. The gradient delay volume of the HPLCsystem was approximately 4 mL and the flow rate was 0.5 mL/min.

[0086] tPA from another commercial source was made up to manufacturer'sspecifications to provide a 1 mg/mL solution. Coagulation Standard(Coagulation Control Level 1 from Sigma Diagnostics, Catalog #C-7916),lyophilized human plasma, was reconstituted according to themanufacturer's instructions. This solution was diluted 10:1 to obtain asolution having roughly 10 times the absorbance of the tPA solution.

[0087] In the test shown in FIG. 1, a sample of pure tPA (25 μL of 1mg/mL tPA) was run over the #109 CMTI derivative-containing column andeluted as described above. The chromatogram shows sharp elution of about90% of the tPA material after about 15 min. In the test shown in FIG. 2,a sample of Coagulation Standard (10× dilution) was run over the #109CMTI derivative-containing column with elution conditions as describedabove. Virtually all the material eluted immediately from the column(was not retained). In the test separation shown in FIGS. 3 and 4, asample containing tPA added to human plasma standard was loaded onto thecolumn and eluted as described above. As can be seen in FIGS. 3 and 4,the tPA was retained and the plasma proteins eluted in the void volume.Bound tPA was released at about 15.4 minutes. It was estimated that thetPA was released at about pH 4. The tPA peak was collected and examinedusing a silver-stained, reducing SDS-polyacrylamide gel, and, whencompared with the starting material, was found to be >95% pure.

EXAMPLE 2

[0088] The tPA affinity ligands isolated from the CMTI library wereexamined further in order to design additional candidate domains thatmight bind to tPA as well.

[0089] As noted above, almost all of the variegation in amino acidpositions used in building the CMTI library occurred between twocyteines at positions 3 and 10 of CMTI-I (see Table 3). In the parentalCMTI protein, these cysteines form disulfide bonds with other cysteineresidues elsewhere in the protein (see SEQ ID NO: 1), however with thesuccessful isolation of affinity ligands from the CMTI library, asecondary library was conceptualized which was based on variegating atruncated 15-amino acid segment of the isolate #109 (see amino acids1-15 of SEQ ID NO: 12). If the C₃ and C₁₀ cysteines of these membersformed a disulfide bond, then a constrained loop having tPA bindingproperties might be obtained. Initial studies with the 15-amino acidsegment derived from affinity ligand isolate #109 bound to achromatographic support indicated that the C₃-C₁₀ loop formed and thatthe immobilized loop bound to tPA.

[0090] The foregoing experiments point to two new families of tPAaffinity ligands isolated in accordance with this invention, comprisingpolypeptides including the sequences:Arg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈-Lys-Asp-Ser-Asp-Cys-Leu-Ala-Glu-Cys-Val-Cys-Leu-Glu-(SEQ ID NO:42) His-Gly-Tyr-Cys-Gly andArg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈, (SEQ ID NO:43)

[0091] wherein X₁ is Trp or Leu; X₂ is Pro, Ser, Thr or Ile; X₃ is Arg,Lys or Thr; X₄ is Ser, Tyr, Thr or Ala; X₅ is Ser, Tyr, Asp, Val, Pro,Ala, His, Asn or Thr; X₆ is Leu, Met, Gn, Arg or Lys; X₇ is Glu, Gly orArg; and X₈ is at least Lys or Met. Since the presence of Met residuesat position 11 in the sequence was not planned but turned out to befavored for binding to tPA, it is likely that other amino acids, forinstance other non-polar amino acids such as Ala, Val, Leu, He, Phe, Proor Trp substituted at position 11 will provide additional tPA-bindinganalogues.

[0092] Following the foregoing description, the characteristic importantfor the separation of tPA from any feed stream can be engineered intothe binding domains of a designed library, so that the method of thisinvention invariably leads to several affinity ligand candidatessuitable for separation of the tPA under desirable conditions of bindingand release. High yield of the tPA without inactivation or disruption ofthe product, with high purity, with the elimination of even closedlyrelated impurities, at acceptable cost and with re-usable or recyclablematerials all can be achieved according to the present invention.Additional embodiments of the invention and alternative methods adaptedto a particular tPA form or feed stream will be evident from studyingthe foregoing description. All such embodiments and alternatives areintended to be within the scope of this invention, as defined by theclaims that follow.

References

[0093] Boschetti, E., J. Chromatography, A 658: 207-236 (1994).

[0094] Ladner, R. C., “Constrained peptides as binding entities,” Trendsin Biotechnology, 13(10): 426-430 (1995).

[0095] Markland, W., Roberts, B. L., Ladner, R. C., “Selection forProtease Inhibitors Using Bacteriophage Display,” Methods in Enzymology,267: 28-51 (1996).

[0096] Narayanan, S. R., “Preparative affinity chromatography ofproteins,” J. Chrom. A, 658: 237-258 (1994).

[0097] Knight, P., BioTechnology, 8: 200 (1990).

[0098] Vedvick, T., Buckholtz, R. G., Engel, M., Urcam, M., Kinney, S.,Provow, S., Siegel, R. S., and Thill, G. P., “High level secretion ofbiologically active aprotinin from the yeast Pichia pastoris”, J.Industrial Microbiol., 7: 197-202 (1991).

[0099] Wagner, S. L., Siegel, R. S., Vedvick, T. S., Raschke, W. C., andVan Nostrand, W. E., “High level expression, purification, andcharacterization of the Kunitz-type protease inhibitor domain ofprotease Nexin-2/amyloid β-protein precursor,” Biochem. Biphys. Res.Comm., 186: 1138-1145 (1992).

1 43 1 29 PRT Cucurbita maxima 1 Arg Val Cys Pro Arg Ile Leu Met Glu CysLys Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu HisGly Tyr Cys Gly 20 25 2 50 PRT Artificial Sequence CMTI-I Library design2 Phe Tyr Ser Gly Ala Arg Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys 1 5 1015 Lys Asp Ser Asp Cys Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr 20 2530 Cys Gly Ala Gly Pro Ser Tyr Ile Glu Gly Arg Ile Val Gly Ser Ala 35 4045 Ala Glu 50 3 150 DNA Artificial Sequence coding sequence for CMTI-ILibrary 3 ttctattccg gagcccgtnn gtgtnntana nntnntnngr rgtgtaagaaggattctgat 60 tgcttagcag aatgcgtttg cctcgagcat ggttattgtg gcgccggtccttcatacatt 120 gaaggtcgta ttgtcggtag cgccgctgaa 150 4 29 PRT ArtificialSequence tPA binding peptide 4 Arg Trp Cys Pro Lys Thr Ser Leu Gly CysMet Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu HisGly Tyr Cys Gly 20 25 5 29 PRT Artificial Sequence tPA binding peptide 5Arg Leu Cys Pro Lys Thr Tyr Leu Gly Cys Met Lys Asp Ser Asp Cys 1 5 1015 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 6 29 PRTArtificial Sequence tPA binding peptide 6 Arg Trp Cys Ser Thr Tyr SerLeu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 7 29 PRT Artificial Sequence tPAbinding peptide 7 Arg Trp Cys Ser Thr Tyr Ser Leu Gly Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 8 29 PRT Artificial Sequence tPA binding peptide 8 Arg Leu CysPro Lys Thr Ser Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu AlaGlu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 9 29 PRT ArtificialSequence tPA binding peptide 9 Arg Trp Cys Ser Thr Tyr Ser Leu Gly CysMet Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu HisGly Tyr Cys Gly 20 25 10 29 PRT Artificial Sequence tPA binding peptide10 Arg Leu Cys Pro Lys Thr Ser Leu Glu Cys Met Lys Asp Ser Asp Cys 1 510 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 11 29PRT Artificial Sequence tPA binding peptide 11 Arg Trp Cys Ser Lys SerSer Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys ValCys Leu Glu His Gly Tyr Cys Gly 20 25 12 29 PRT Artificial Sequence tPAbinding peptide 12 Arg Leu Cys Pro Lys Thr Asp Leu Gly Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 13 29 PRT Artificial Sequence tPA binding peptide 13 Arg TrpCys Pro Lys Ser Ser Met Gly Cys Lys Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 14 29 PRTArtificial Sequence tPA binding peptide 14 Arg Trp Cys Pro Arg Thr ValGln Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 15 29 PRT Artificial Sequence tPAbinding peptide 15 Arg Trp Cys Pro Thr Ala Pro Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 16 29 PRT Artificial Sequence tPA binding peptide 16 Arg LeuCys Pro Lys Thr Asp Leu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 17 29 PRTArtificial Sequence tPA binding peptide 17 Arg Trp Cys Pro Lys Ser AlaLeu Asp Cys Lys Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 18 29 PRT Artificial Sequence tPAbinding peptide 18 Arg Trp Cys Thr Lys Thr Ser Arg Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 19 29 PRT Artificial Sequence tPA binding peptide 19 Arg TrpCys Ile Arg Thr Asp Leu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 20 29 PRTArtificial Sequence tPA binding peptide 20 Arg Trp Cys Pro Lys Thr SerLeu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 21 29 PRT Artificial Sequence tPAbinding peptide 21 Arg Trp Cys Pro Arg Thr Val Arg Arg Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 22 29 PRT Artificial Sequence tPA binding peptide 22 Arg TrpCys Pro Lys Thr His Lys Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 23 29 PRTArtificial Sequence tPA binding peptide 23 Arg Trp Cys Pro Lys Thr SerLeu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 24 29 PRT Artificial Sequence tPAbinding peptide 24 Arg Trp Cys Pro Lys Ser Thr Leu Gly Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 25 29 PRT Artificial Sequence tPA binding peptide 25 Arg TrpCys Pro Lys Ser Thr Leu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 26 29 PRTArtificial Sequence tPA binding peptide 26 Arg Trp Cys Pro Lys Tyr ThrLeu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 27 29 PRT Artificial Sequence tPAbinding peptide 27 Arg Trp Cys Pro Arg Ser Ser Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 28 29 PRT Artificial Sequence tPA binding peptide 28 Arg TrpCys Pro Lys Tyr Thr Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 29 29 PRTArtificial Sequence tPA binding peptide 29 Arg Trp Cys Pro Arg Ser AsnLeu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 30 29 PRT Artificial Sequence tPAbinding peptide 30 Arg Trp Cys Pro Arg Ser Asn Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 31 29 PRT Artificial Sequence tPA binding peptide 31 Arg TrpCys Pro Lys Tyr Thr Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 32 29 PRTArtificial Sequence tPA binding peptide 32 Arg Trp Cys Pro Lys Tyr ThrLeu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 33 29 PRT Artificial Sequence tPAbinding peptide 33 Arg Trp Cys Pro Lys Tyr Thr Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 34 29 PRT Artificial Sequence tPA binding peptide 34 Arg TrpCys Pro Arg Ser Thr Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 35 29 PRTArtificial Sequence tPA binding peptide 35 Arg Trp Cys Pro Lys Thr SerLeu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 36 29 PRT Artificial Sequence tPAbinding peptide 36 Arg Trp Cys Pro Lys Tyr Thr Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 37 29 PRT Artificial Sequence tPA binding peptide 37 Arg TrpCys Pro Arg Ser Ser Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 38 29 PRTArtificial Sequence tPA binding peptide 38 Arg Trp Cys Pro Lys Ser ThrLeu Gly Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 39 29 PRT Artificial Sequence tPAbinding peptide 39 Arg Trp Cys Pro Arg Ser Ser Leu Glu Cys Met Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 40 29 PRT Artificial Sequence tPA binding peptide 40 Arg TrpCys Pro Arg Ser Asn Leu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 LeuAla Glu Cys Val Cys Leu Glu His Gly Tyr Cys Gly 20 25 41 29 PRTArtificial Sequence tPA binding peptide 41 Arg Trp Cys Pro Arg Ser AsnLeu Glu Cys Met Lys Asp Ser Asp Cys 1 5 10 15 Leu Ala Glu Cys Val CysLeu Glu His Gly Tyr Cys Gly 20 25 42 29 PRT Artificial Sequence tPAbinding peptide 42 Arg Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Lys AspSer Asp Cys 1 5 10 15 Leu Ala Glu Cys Val Cys Leu Glu His Gly Tyr CysGly 20 25 43 11 PRT Artificial Sequence tPA binding peptide 43 Arg XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10

1. An affinity ligand suitable for isolating tPA from a solutioncontaining it comprising a polypeptide including the amino acidsequence: Arg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈, wherein X₁ Trp or Leu; X₂is Pro, Ser, Thr or Me, X₃ is Arg, Lys or Thr; X₄ is Ser, Tyr, Thr orAla; X₅ is Ser, Tyr, Asp, Val, Pro, Ala, His, Asn or Thr; X₆ is Leu,Met, Gins Arg or Lys; X₇ is Glu, Gly or Arg, and X₈ is Lys, Met, Ala,Val, Leu, lie, Phe, Pro or Trp.
 2. An affinity ligand suitable forisolating tPA from a solution containing it comprising a polypeptideincluding the amino acid sequence:Arg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈-Lys-Asp-Ser-Asp-Cys-Leu-Ala-Glu-Cys-Val-Cys-Leu-Glu-His-Gly-Tyr-Cys-Gly,wherein X₁ is Trp or Leu; X₂ is Pro, Se. Thr or Ile; X₃ is Arg, Lys orThr; X₄ is Ser, Tyr, Thr or Ala; X₅ is Ser, Tyr, Asp, Val, Pro, Ala,His, Asn or Tr; X₆ is Leu, Met, Gin, Arg or Lys; X₇ is Glu, Gly or Arg;and X₈ is Lys, Met, Ala, Val, Leu, lie, Phe, Pro or Trp.
 3. An affinityligand suitable for isolating tPA from a solution containing it havingan amino acid sequence including an amino acid sequence selected fromthe group consisting of: RLCPKTDLGCMK; RLCPKTDLGCMKDSDCLAECVCLEHGYCG;RLCPKTDLGCMKDSDCLAECVCLEHGYCGA; and EARLCPKTDLGCMKDSDCLAECVCLEHGYCGA.


4. An affinity ligand capable of binding to tPA in a solution at pH 7and dissociating from tPA at a pH of 5 or lower.
 5. An affinity ligandaccording to claim 4 including an amino acid sequence selected from thegroup consisting of: Arg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈, wherein X₁ isTrp or Leu; X₂ is Pro, Ser, Thr or Ile; X₃ is Arg, Lys or Thr; X₄ isSer, Tyr, Thr or Ala; X₅ is Ser, Tyr, Asp, Val, Pro, Ala, His, Asn orThr; X₆ is Leu, Met, Gin, Arg or Lys; X₇ is Glu, Gly or Arg; and X₈ isLys, Met, Ala, Val, Leu, Ile, Phe, Pro or Trp; andArg-X₁-Cys-X₂-X₃-X₄-X₅-X₆-X₇-Cys-X₈-Lys-Asp-Ser-Asp-Cys-Leu-Ala-Glu-Cys-Val-Cys-Leu-Glu-His-Gly-Tyr-Cys-Gly,wherein X₁ is Trp or Leu; X₂ is Pro, Ser, Thr or Ile; X₃ is Arg, Lys orThr; X₄ is Ser, Tyr, Thr or Ala; X₅ is Ser, Tyr, Asp, Val, Pro, Ala,His, Asn or Thr; X₆ is Leu, Met, Gln, Arg or Lys; X₇ is Glu, Gly or Arg;and X₈ is Lys, Met, Ala, Val, Leu, Ile, Phe, Pro or Trp.
 6. An affinityligand according to claim 4, wherein the affinity ligand has an aminoacid sequence including an amino acid sequence selected from the groupconsisting of: RWCPKTSLGCMKDSDCLAECVCLEHGYCGRLCPKTYLGCMKDSDCLAECVCLEHGYCG RWCSTYSLGCMKDSDCLAECVCLEHGYCGRWCSTYSLGCMKDSDCLAECVCLEHGYCG RLCPKTSLECMKDSDCLAECVCLEHGYCGRWCSTYSLGCMKDSDCLAECVCLEHGYCG RLCPKTSLECMKDSDCLAECVCLEHGYCGRWCSKSSLECMKDSDCLAECVCLEHGYCG RLCPKTDLGCMKDSDCLAECVCLEHGYCGRWCPKSSMGCKKDSDCLAECVCLEHGYCG RWCPRTVQECMKDSDCLAECVCLEHGYCGRWCPTAPLECMKDSDCLAECVCLEHGYCG RLCPKTDLGCMKDSDCLAECVCLEHGYCG RWCPKSALDCKKDSDCLAECVCLEHGYCG RWCTKTSRECMKDSDCLAECVCLEHGYCGRWCLRTDLGCMKDSDCLAECVCLEHGYCG RWCPKTSLGCMKDSDCLAECVCLEHGYCGRWCPRTVRRCMKDSDCLAECVCLEHGYCG RWCPKTHKEC MKDSDCLAECVCLEHGYCGRWCPKTSLECMKDSDCLAECVCLEHGYCG RWCPKSTLGCMKDSDCLAECVCLEHGYCGRWCPKSTLGCMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSNLECMKDSDCLAECVCLEHGYCG RWCPRSNLECMKDSDCLAECVCLEHGYCGRWCPKYTLECMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPKYTLECMKDSDCLAECVCLEHGYCG RWCPRSTLECMKDSDCLAECVCLEHGYCGRWCPKTSLGCMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPKSTL GCMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPRSNLECMKDSDCLAECVCLEHGYCG andRWCPRSNLECMKDSDCLAECVCLEHGYCG.


7. An affinity ligand according to claim 4 including an amino acidsequence selected from the group consisting of: RLCPKTDLGCMK;RLCPKTDLGCMKDSDCLAECVCLEHGYCG; RLCPKTDLGCMKDSDCLAECVCLEHGYCGA; andEARLCPKTDLGCMKDSDCLAECVCLEHGYCGA.


8. A method for isolating an affinity ligand suitable for separating tPAfrom a solution containing it, the method comprising: (a) selecting afirst solution condition at which it is desired that an affinity ligandshould bind to the tPA; (b) selecting a second solution condition atwhich it is desired that an affinity complex between the tPA and theaffinity ligand will dissociate, wherein the second solution conditionis different from the first solution condition; (c) providing a libraryof analogues of a candidate binding domain, wherein each analoguediffers from said candidate binding domain by variation of the aminoacid sequence at one or more amino acid positions within the domain; (d)contacting said library of analogues with tPA at the first solutioncondition, for sufficient time to permit analogue/tPA binding complexesto form; (e) removing analogues that do not bind under the firstsolution condition; (f) altering the conditions of the solution ofcontacting step (e) to the second solution condition; and (g) recoveringthe candidate binding analogues released under the second solutioncondition, wherein the recovered analogues identify isolated tPAaffinity ligands.
 9. A method for isolating an affinity ligand suitablefor separating tPA from a solution containing it, the method comprising:(a) preparing a library of analogues of Cucurbida maxima trypsininhibitor-I (SEQ ID NO. 1) by making amino acid substitutions at aminoacid positions 2, 4, 5, 6, 7, 8, 9 and 11; (b) contacting said libraryof analogues with immobilized tPA under conditions permitting formationof analogue/tPA binding complexes and at pH 7 or higher; (c) removinganalogues that do not bind tPA under the conditions of said contactingstep; (d) altering the conditions of the contacting step by lowering thepH to pH 5 or lower; and (e) recovering the analogues released at pH 5or lower.
 10. A method for purifying tPA from a solution containing tPAcomprising: (a) immobilizing a polypeptide including the amino acidsequence of SEQ ID NO: 42 on a chromatographic support; (b) contacting asolution containing tPA with said support at pH 7 or above; (c) removingother components of said solution from contact with said support; (d)recovering tPA from said support at pH 5 or lower.
 11. A method forpurifying tPA from a solution containing tPA comprising: (a)immobilizing an affinity ligand according to claim 2 on achromatographic support; (b) contacting a solution containing tPA withsaid support at pH 7 or above; (c) removing other components of saidsolution from contact with said support; (d) recovering tPA from saidsupport at pH 5 or lower.
 12. A method according to claim 9, whereinsaid affinity ligand has an amino acid sequence including an amino acidsequence selected from the group consisting of RLCPKTDLGCMK;RLCPKTDLGCMKDSDCLAECVCLEHGYCG; RLCPKTDLGCMKDSDCLAECVCLEHGYCGA; andEARLCPKTDLGCMKDSDCLAECVCLEHGYCGA.


13. A method according to claim 10, wherein said affinity ligand has anamino acid sequence including an amino acid sequence selected from thegroup consisting of: RWCPKTSLGCMKDSDCLAECVCLEHGYCGRLCPKTYLGCMKDSDCLAECVCLEHGYCG RWCSTYSLGCMKDSDCLAECVCLEHGYCGRWCSTYSLGCMKDSDCLAECVCLEHGYCG RLCPKTSLECMKDSDCLAECVCLEHGYCGRWCSTYSLGCMKDSDCLAECVCLEHGYCG RLCPKTSLECMKDSDCLAECVCLEHGYCGRWCSKSSLECMKDSDCLAECVCLEHGYCG RLCPKTDLGCMKDSDCLAECVCLEHGYCGRWCPKSSMGCKKDSDCLAECVCLEHGYCG RWCPRTVQECMKDSDCLAECVCLEHGYCGRWCPTAPLECMKDSDCLAECVCLEHGYCG RLCPKTDLGCMKDSDCLAECVCLEHGYCG RWCPKSALDCKKDSDCLAECVCLEHGYCG RWCTKTSRECMKDSDCLAECVCLEHGYCGRWCLRTDLGCMKDSDCLAECVCLEHGYCG RWCPKTSLGCMKDSDCLAECVCLEHGYCGRWCPRTVRRCMKDSDCLAECVCLEHGYCG RWCPKTHKECMKDSDCLAECVCLEHGYCGRWCPKTSLECMKDSDCLAECVCLEHGYCG RWCPKSTLGCMKDSDCLAECVCLEHGYCGRWCPKSTLGCMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSNLECMKDSDCLAECVCLEHGYCG RWCPRSNLECMKDSDCLAECVCLEHGYCGRWCPKYTLECMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPKYTLECMKDSDCLAECVCLEHGYCG RWCPRSTLECMKDSDCLAECVCLEHGYCGRWCPKTSLGCMKDSDCLAECVCLEHGYCG RWCPKYTLECMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPKSTLGCMKDSDCLAECVCLEHGYCGRWCPRSSLECMKDSDCLAECVCLEHGYCG RWCPRSNLECMKDSDCLAECVCLEHGYCG andRWCPRSNLECMKDSDCLAECVCLEHGYCG.


14. A method according to claim 10, wherein said affinity ligand has anamino acid sequence including an amino acid sequence selected from thegroup consisting of: RWCPKTSLGCMK; RWCPKTSLGCMKDSDCLAECVCLEHGYCG;RWCPKTSLGCMKDSDCLAECVCLEHGYCGA; EARWCPKTSLGCMKDSDCLAECVCLEHGYCGA;RLCPKTDLGCMK; RLCPKTDLGCMKDSDCLAECVCLEHGYCG;RLCPKTDLGCMKDSDCLAECVCLEHGYCGA; and EARLCPKTDLGCMKDSDCLAECVCLEHGYCGA.